Amyloid-beta-binding peptides and the use thereof for the treatment and diagnosis of Alzheimer&#39;s disease

ABSTRACT

The invention relates to amyloid beta-binding peptides and the use thereof for the treatment and the diagnosis of Alzheimer&#39;s disease.

REFERENCE TO RELATED APPLICATION

This is a continuation application of Ser. No. 15/022,749, filed Mar.17, 2016, currently pending. The subject matter of the aforementionedprior applications is hereby incorporated herein by reference.

The invention relates to amyloid beta-binding peptides and the usethereof for the treatment and the diagnosis of Alzheimer's disease.

PRIOR ART

Multivalent amyloid beta-binding polymeric substances, composed ofmultiple interconnected substances that on their own already haveamyloid beta-binding properties, and the use of these substances, inparticular in medicine, are known from the unexamined patent applicationWO 2013/150127 A2.

Given the demographic trend over the coming decades, the number ofindividuals suffering from age-related diseases will rise. In thisregard, especially so-called Alzheimer's disease (AD, Latin: MorbusAlzheimer) shall be mentioned.

So far, no active ingredient or drug exists that acts against the causesof AD. The previously used and approved drugs alleviate some of thesymptoms that occur with Alzheimer's disease. However, they are not ableto slow the progression of the disease or provide a cure. Severalsubstances exist that, in animal experiments, achieved successes interms of prevention, but not (necessarily) in the treatment of AD.Active ingredients against neurodegenerative diseases are known from DE10 2006 015 140 A1.

One trait of Alzheimer's disease is extracellular deposits of theamyloid beta peptide (A beta peptide, Aß, or Aß peptide). These depositsof the A beta peptide in plaque are typically found in the brains of ADpatients post mortem. This is the reason why various forms of the A betapeptide, such as fibrils, are considered to be responsible for thedevelopment and progression of the diseases. Additionally, for someyears now, the small, freely diffusable A beta oligomers have beenregarded as the primary causative factor in the development andprogression of AD.

The substances known from the prior art reduce the concentration of Abeta monomers and/or oligomers in a wide variety of ways. For example,gamma-secretase modulators are known, which were used for prevention inanimal experiments.

Various sequences of D-amino acids that bind to A beta peptides areknown from WO 02/081505 A2. These sequences of D-amino acids bind toamyloid beta peptides with a dissociation constant (K_(D) value) of 4μM.

Hybrid compounds that are composed of aminopyrazoles and peptides andprevent A beta oligomerization are known from WO 2011/147797 A2.

Compounds that interact with A beta peptides are known from DE 10 2008037 564 A1, DE 696 21 607 T2 or DE 10 2010 019 336 A1. The binding of amultivalent polymer to two binding partners is described in WO2008/116293 A1.

In the case of many substances that showed positive results in animalexperiments, this effect could not be confirmed in clinical humanstudies. In phase II and III clinical studies, only individuals who havebeen unambiguously diagnosed with AD are allowed to be treated. A minorreduction in the A beta monomer concentration will not be sufficient toprevent existing A beta oligomers from multiplying, such as by way of aprion-like mechanism. The multiplication of A beta oligomers or, evenbetter, destruction of the same or rendering these ineffective, however,is absolutely necessary to influence the progression of the disease.

Until now, Alzheimer's disease is primarily diagnosed by way ofneuropsychological tests, by conducting experiments on persons in whichsymptoms of dementia were detected. However, it is known that A betaoligomers, and the fibrils and plaque ensuing subsequently, develop inthe brain of patients up to 20 years prior to the appearance of symptomsand can already have caused irreversible damage. However, to date, it isnot possible yet to diagnose AD before symptoms appear.

As a result, there is a continued need for new compounds (activeingredients) that bind very specifically and with high affinity to Abeta oligomers, and thereby prevent the multiplication thereof. Thesecompounds should not produce undesirable side effects, and in particularcause no immune response. The compounds should additionally detect evensmall concentrations of toxic A beta oligomers, and thus also the smallfreely diffusable oligomers and completely destroy and/or prevent the(prion-like) multiplication of the same.

Furthermore, there is also a need for new compounds that can be used asprobes for detecting and marking A beta oligomers, in particular whenthese oligomers occur only in small concentrations.

SUMMARY OF THE INVENTION

It is thus the object of the invention to provide compounds for

A) the causal treatment of Alzheimer's disease by these compoundspreventing the formation of toxic amyloid beta oligomers, fibrils oraggregates, or by eliminating oligomers, fibrils or aggregates that havealready developed, or by causing the detoxification of the same;

B) allowing Alzheimer's disease to be diagnosed by being able to usethese compounds as probes for in vivo imaging.

This covers additional objects, such as the use of the compounds inmedicine.

It is also an object of the invention to provide novel peptides,preferably derivatives of the D-enantiomeric D-peptide D3, which havemore efficient properties than D3. The properties include, among otherthings, a higher binding affinity and specificity for A beta species,inhibition of A beta fibril formation, inhibition of A betacytotoxicity, elimination or detoxification of A beta oligomers, fibrilsand other aggregates, and conversion of A beta amyloid fibrils,protofibrils or oligomers into non-toxic, non-amyloidogenic species.

Hereafter, the terms “A beta,” “amyloid beta,” “amyloid ß” and “Aß” areused synonymously.

Solution to the Problem

The object is achieved by the peptide, the kit, and the compositionaccording to the main claims, and by the methods and uses according tothe additional independent claims.

Advantageous embodiments will be apparent from the respective claimsdependent thereon.

DESCRIPTION OF THE INVENTION

A peptide according to the invention is a peptide comprising at leastone amino acid sequence that binds to amyloid beta species, with thisproperty being either preserved or amplified, and in which the freeC-terminus, which is to say the C-terminal carboxyl group, is modifiedso that the C-terminus does not have a negative charge, but instead isneutral or has one or more positive charges.

The peptides according to the invention also include those peptidescomprising at least one peptide binding to an amyloid-beta species,wherein the peptide includes a linear amino acid sequence which enablesit to bind to A-beta, with this property being either preserved oramplified by the peptide being present in cyclized form as a result of acovalent bond of the two ends thereof.

The peptides according to the invention particularly advantageously alsoachieve the object by providing a peptide having no negative charge atthe C-terminus. This advantageously causes the peptide to be able tobind with higher affinity to the target molecule as compared to apeptide comprising a carboxyl group at the free C-terminus. Peptideshaving a free, non-modified carboxyl group have a negative charge atthis end in the physiological state.

In one embodiment of the invention, the peptides according to theinvention are modified in the physiological state, in particular at pH 6to 8, in particular 6.5 to 7.5, in particular at pH 6.0, pH 6.1, pH 6.2,pH 6.3, pH 6.4, pH 6.5, pH 6.6, pH 6.7, pH 6.8, pH 6.9, pH 7.0, pH 7.1,pH 7.2, pH 7.3, pH 7.4, pH 7.5, pH 7.6, pH 7.7, pH 7.8, pH 7.9 or pH8.0, so that the C-terminus does not carry a negative charge, butinstead is neutral or has one or more positive charges.

In one embodiment of the invention, the peptide is characterized in thatan acid amide group is present at the free C-terminus in place of thecarboxyl group. Instead of the carboxyl group (—COOH group), an acidamide group (—CONH₂ group) is thus provided at the C-terminus.

The peptide is thus particularly advantageously amidated at the freeC-terminus.

This particularly advantageously achieves the further object that apeptide having no excess negative charge is present, which can bind withhigher affinity to the target molecule and can be obtained in a simplemanner.

In a further embodiment of the invention, the following further groupsare present in place of the carboxyl group: COH, COCl, COBr, CONH-alkylgroup, CONH-alkyl-amine group (positive net charge), and so forth,wherein there is no limitation to these, provided the technical teachingof the main claim is followed.

This object is achieved in particular by a peptide containing an aminoacid sequence according to SEQ ID NO: 1 (RD2), SEQ ID NO: 2 (D3), SEQ IDNO: 3 (DB3), SEQ ID NO: 9 (D3r), SEQ ID NO: 10 (D3p), SEQ ID NO: 11(D3a) and/or SEQ ID NO: 12 (D3p2k) and/or homologs, fragments, and partsthereof. These peptides are substance units (hereafter often referred toas “monomers”) that bind to amyloid-beta species.

In one variant of the invention, monomers are used which bind to an Abeta monomer and/or A beta oligomers and/or fibrils of the A betapeptide with a dissociation constant (Ko value) of no more than 500 μM,preferably 250, 100, 50 μM, particularly preferably 25, 10, 6 μM, and inparticular 4, 2, 1 μM or sub-μM.

The object is, in particular, also achieved by polymers composed of twoor more of the above-mentioned monomers or peptides according to theinvention, in particular dimers of SEQ ID NO: 4 (RD2D3), SEQ ID NO: 5(D3RD2), SEQ ID NO: 6 (D3D3), SEQ ID NO: 7 (RD2RD2) and/or SEQ ID NO: 8(DB3DB3) and/or homologs thereof. The dimers are composed of two monomerunits, which each bind to amyloid-beta species.

The polymers according to the invention, composed of monomers that, inturn, bind to A beta oligomers, exhibit clear, synergistic effects withrespect to the selectivity and affinity thereof to the A beta oligomers,compared to the monomers. In other words, the polymers according to theinvention, in particular the dimers, selected from the group consistingof SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and/or SEQ IDNO: 8, are superior to the monomers of which they are composed.Synergistic effects within the meaning of the present invention areeffects that exhibit a higher selectivity and/or affinity with respectto relevant A beta species, in particular the K_(D) value regarding thebinding to A beta species compared to the individual monomer units.

In a further particularly advantageous embodiment of the invention, thepolymers, and more particularly the dimers, (in vitro or in vivo)advantageously act more efficiently than the monomers in the animalmodel experiment.

In one variant of the invention, polymers are used which bind to an Abeta monomer and/or A beta oligomers and/or fibrils of the A betapeptide, with a dissociation constant (K_(D) value) of no more than 500μM, preferably 250, 100, 50 μM, particularly preferably 25, 10, 1 μM,particularly preferably with a dissociation constant (K_(D) value) of nomore than 500 nM, 250, 100, 50, particularly preferably 25, 10, 1 nM,500 μM, 100, 50, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 μM to sub-μM,wherein any intermediate value is possible.

In one embodiment of the invention, the affinity is defined by way ofthe dissociation constant (K_(D) value).

In an advantageous embodiment of the invention, the dissociationconstant (K_(D) value) of the peptide according to the invention isadvantageously reduced compared to linear, binding peptides in which thefree C-terminus, which is to say the C-terminal carboxyl group, has anegative charge. This is associated with improved properties of thepeptides according to the invention, such as higher binding affinity andhigher effectiveness in the decomposition and/or the prevention of theformation of toxic amyloid beta species. This applies in particular, butnot exclusively, to a low K_(D) value at the high-affinity site of the Abeta (monomer, oligomer, and fibrils).

Fragments and parts advantageously exhibit a similar or identical effectas the peptides according to the invention.

In one variant of the invention, the peptides according to theinvention, and more particularly the peptides according to SEQ ID NO: 1,SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6,SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11and/or SEQ ID NO: 12 and the homologs, fragments or parts thereof, aresubstantially, preferably at least 50%, 60%, 75%, 80%, particularlypreferably 85%, 90%, 95%, and in particular 96%, 97%, 98%, 99%, 100%composed of D-enantiomeric amino acids.

A polymer within the meaning of the invention is formed of 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more monomersthat bind to amyloid beta species, and in particular to oligomers.

The polymer is in particular made of monomers selected from the groupconsisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 9,SEQ ID NO: 10, SEQ ID NO: 11 and/or SEQ ID NO: 12.

In one embodiment of the invention, the polymer may be selected from thegroup consisting of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO:7 and/or SEQ ID NO: 8, and the homologs thereof, which on their ownalready bind to amyloid beta species.

In one embodiment of the invention, the peptides according to SEQ IDNOS: 1-12 are composed of D-enantiomeric amino acids.

In one embodiment of the invention, the peptides according to SEQ IDNOS: 1-12 are provided with an acid amide group at the free C-terminus.Monomers, such as D3, DB3 or RD2 according to SEQ ID NOS: 1-3, or D3r,Drp, D3a or D3(p2k) according to SEQ ID NOS: 9-12 are then amidated atposition 12 at the free C-terminus. Polymers, such as RD2D3, D3RD2,D3D3, RD2RD2 or DB3DB3, are amidated at position 24 at the freeC-terminus.

In a further embodiment of the invention, the peptides according to SEQID NOS: 1-12 are covalently bonded to each other at the free C-terminusand the free N-terminus and are then present in corresponding cyclizedform. The ring closure also advantageously causes the carboxyl group atthe free C-terminus to no longer be present.

The peptide according to the invention advantageously comprises an aminoacid sequence in which the cyclization of the linear molecule has beencarried out, for example, by way of a covalent bond of the first aminoacid with the last amino acid, such as by way of a condensationreaction. Other options for cyclization exist, of course, for example bycoupling other amino acids to each other. The coupling of the secondamino acid to the last amino acid shall only be mentioned by way ofexample. Any possible other coupling is equally conceivable.

If the first and last amino acids of the peptide are coupled to eachother, this advantageously results in no open ends being present in thepeptide chain (amino acid sequence).

A further result of this measure is that all peptides having linearamino acid sequences that, after cyclization, yield the same, no longerdistinguishable amino acid order, are identical in this regard.

Example

The linear amino acid sequence of the known peptide D3 is rprtrlhthrnr.The corresponding cyclized peptide “cD3” linked between the N-terminalamino group and the C-terminal carboxyl group by an amide bond is nolonger distinguishable from the cyclized peptides prtrlhthrnrr,rtrlhthrnrrp, trlhthrnrrpr, rlhthrnrrprt, lhthrnrrprtr, hthrnrrprtrl,thrnrrprtrlh, hrnrrprtrlht, rnrrprtrlhth, nrrprtrlhthr, or rrprtrlhthrn.It is still possible to derive the cD3 from each of these sequences.

The effects claimed according to the invention of higher affinity andeffectiveness moreover occur with respect to one, preferably even withrespect to each, linear binding peptide from which a cyclized orotherwise modified peptide according to the invention can be derived.

Otherwise, the production of cyclized peptides is state of the art andcan be carried out, for example, according to the methods as describedin DE 102005049537 A1.

The cyclization via the first and last amino acids of the peptideadvantageously also means that there are no longer any “open” ends ofthe peptide chain, which often represent points of attack forpeptide-decomposing activities in cells, animals or humans, such as byway of aminopeptidases and carboxypeptidases.

Using cyclized monomers according to the invention, such as cD3, cRD2and so forth, or cyclized polymers, such as cRD2D3, cD3D3, cDB3DB3 andso forth, also advantageously achieves that, as a side effect, thesecyclized peptides according to the invention are also potentially noteasily degraded, although this effect is not decisive. Incidentally, aswas shown, this effect also applies only for the case of a head-to-tailor tail-to-head cyclization, in which the two ends of the linear peptideare correspondingly coupled to each other.

In a further embodiment of the invention, the polymers are composed ofidentical monomers, such as D3, RD2 or DB3, or of a combination of 2, 3,4, 5, 6, 7, 8, 9 or 10 different, varying monomers as mentioned above,as so-called combination polymers. Some of the monomers can also beidentical. The number of identical monomers in the combination polymersis freely selectable.

Polymers can be produced by chemical synthesis or peptide synthesis, forexample.

In one embodiment of the invention, the monomers are covalently bondedto each other. In a further embodiment, the monomers are not covalentlybonded to each other.

A covalent bond or coupling of the monomer units within the meaning ofthe invention is present if the peptides are linearly coupled to eachother head to head, tail to tail, or head to tail, with or withoutinterposed linkers or linker groups.

A non-covalent bond within the meaning of the invention exists if themonomers are coupled to each other via biotin and streptavidin, inparticular streptavidin tetramer, for example.

In one variant of the present invention, the monomers can be linearlycoupled to each other, in particular as described above. In anothervariant, the monomers are coupled to each other in a branched manner toobtain the polymer according to the invention.

According to the invention, the branched polymer can be a dendrimer inwhich the monomers are covalently or not covalently coupled to eachother.

Alternatively, the monomers can also be coupled to a platform molecule(such as PEG or sugar) and thus form a branched polymer.

Alternatively, combinations of these options are also possible.

Monomers and polymers are hereinafter referred to as peptides accordingto the invention.

In one variant of the invention, a peptide is used having the amino acidsequence according to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ IDNO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ IDNO: 9, SEQ ID NO: 10, SEQ ID NO: 11 and/or SEQ ID NO: 12 and/or homologsthereof having an identity of 50%.

Within the meaning of the invention, “homologous sequences” or“homologs” shall mean that an amino acid sequence has an identity of atleast 50, 55, 60, 65, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81,82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,100% with one of the above-mentioned amino acid sequences of themonomers. Instead of the term “identity,” the terms “homologous” or“homology” are used as synonyms in the present description. The identitybetween two nucleic acid sequences or polypeptide sequences iscalculated by comparison with the aid of the BESTFIT program, based onthe algorithm by Smith, T. F. and Waterman, M. S (Adv. Appl. Math. 2:482-489 (1981)), setting the following parameters for amino acids: Gapcreation penalty: 8 and Gap extension penalty: 2; and setting thefollowing parameters for nucleic acids: Gap creation penalty: 50 and Gapextension penalty: 3. The identity between two nucleic acid sequences orpolypeptide sequences is preferably defined by the identity of thenucleic acid sequence/polypeptide sequence over the entire respectivesequence length, as it is calculated by comparison with the aid of theGAP program, based on the algorithm by Needleman, S. B. and Wunsch, C D.(J. Mol. Biol. 48: 443-453), setting the following parameters for aminoacids: Gap creation penalty: 8 and Gap extension penalty: 2; and settingthe following parameters for nucleic acids: Gap creation penalty: 50 andGap extension penalty: 3.

Two amino acid sequences are identical within the meaning of the presentinvention if they have the same amino acid sequence.

In one variant, homologs shall be understood to mean the correspondingretro-inverse sequences of the above-mentioned monomers. According tothe invention, the term “retro-inverse sequence” denotes an amino acidsequence that is composed of amino acids in the enantiomeric form(inverse: chirality of the alpha carbon atom is inverted), and in whichadditionally the sequence order was reversed compared to the originalamino acid sequence (retro=reverse).

In a further variant, the peptides according to the invention bind toparts of the amyloid beta peptide.

In a further variant, the peptides according to the invention havesequences that differ from the indicated sequences by up to three aminoacids.

Moreover, peptides containing the above-mentioned sequences are alsoused as sequences.

In a further variant, the peptides comprise fragments of theabove-mentioned sequences or comprise homologous sequences with respectto the above-mentioned sequences.

According to the invention, the peptide is a peptide for use inmedicine, and preferably for treating Alzheimer's disease.

In one embodiment of the present invention, the peptide is substantiallycomposed of D-amino acids.

Within the meaning of the present invention, the expression“substantially composed of D-enantiomeric amino acids” shall mean thatat least 50%, 60%, preferably 75%, 80%, particularly preferably 85%,90%, 95%, and in particular 96%, 97%, 98%, 99% or 100% of the monomersto be used according to the invention will be D-enantiomeric aminoacids.

In one embodiment of the present invention, the monomer peptidesaccording to the invention are derivatives of the D-enantiomericD-peptide D3. Derivatives within the meaning of the invention arepeptide sequences that are derived from D3 and are obtained according toone of the three following methods:

a) changing the order and/or number of the amino acid building blocks inD3. Only amino acids that are present in the D3 sequence are used.

d) deleting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 amino acids of the D3sequence.

c) exchanging 1, 3, 4, 5, 6 or 6 amino acids with other amino acids,preferably D-enantiomers.

A further variant concerns a peptide according to the invention forinhibiting the fibril formation of amyloid beta oligomers. The peptidesaccording to the invention detoxify the A beta oligomers or polymersformed thereof, and fibrils, by binding thereto, thus converting theminto non-toxic compounds. The present invention thus also relates to amethod for detoxifying the A beta oligomers, or polymers or fibrilsformed thereof.

In one embodiment, the invention also relates to peptides according tothe invention that are linked to another substance.

The linkage within the meaning of the invention is a chemical bond as itis defined in Römpp Chemie Lexikon (chemistry encyclopedia), 9thedition, volume 1, page 650 et seq., Georg Thieme Verlag, Stuttgart,preferably a principal valence bond, and more particularly a covalentbond.

The substances, in one variant, are pharmaceutical products or activeingredients, defined according to German Drug Act § 2 or § 4 (19), asamended in September 2012. In one alternative, active ingredients aretherapeutically active substances that are used as active pharmaceuticalsubstances. Preferably anti-inflammatory agents are used.

In a further variant, the substances are compounds that enhance theeffect of the peptides.

In one alternative, such compounds are aminopyrazole and/oraminopyrazole derivatives. Aminopyrazole derivatives within the meaningof the invention is 3-aminopyrazole-5-carboxylic acid or3-nitropyrazole-5-carboxylic acid and all derivatives in which theheterocyclic CH group was replaced with —CR— or —N or —O— or —S—, andall peptidic dimers, trimers or tetramers derived therefrom, preferablyaminopyrazole trimer.

In a further alternative, they are compounds that improve the solubilityof the peptides and/or the passage thereof through the blood brainbarrier.

In one alternative, the peptides according to the invention have anyarbitrary combination of at least two or more features of theabove-described variants, embodiments and/or alternatives.

Within the scope of the invention, it was furthermore found that thepeptides modified according to the invention have a higher bindingaffinity to A beta species, and particularly the especially toxicamyloid beta oligomers, than linear binding peptides in which the freeC-terminus, which is to say the C-terminal carboxyl group, is notmodified and correspondingly has a negative charge. This means that theK_(D) value is lower in the peptides according to the invention than inlinear peptides in which the free C-terminus, which is to say theC-terminal carboxyl group, is not modified and correspondingly has anegative charge.

In a further preferred embodiment of the invention, the binding affinityof the peptides modified according to the invention having no negativecharge at the C-terminus, compared to linear peptides having a negativecharge at the C-terminus, but otherwise having the same amino acidsequence, is thus increased by 1%, 2, 3, 4, 5, 6, 7, 8, 9, in particular10%, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81,82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,in particular 100%, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110,111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124,125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138,139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152,153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166,167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180,181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194,195, 196, 197, 198, 199, in particular 200%, 201, 202, 203, 204, 205,206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219,220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233,234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247,248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261,262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275,276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289,290, 291, 292, 293, 294, 295, 296, 297, 298, 299, in particular 300%,301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314,315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328,329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342,343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356,357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370,371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384,385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398,399, in particular 400%, 401, 402, 403, 404, 405, 406, 407, 408, 409,410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423,424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437,438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451,452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465,466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479,480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493,494, 495, 496, 497, 498, 499, advantageously even 500%, 501, 502, 503,504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517,518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531,532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545,546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559,560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573,574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587,588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, particularlyadvantageously 600%, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610,611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624,625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638,639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 652,653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666,667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680,681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694,695, 696, 697, 698, 699, particularly advantageously 700%, 701, 702,703, 704, 705, 706, 707, 708, 709, 710, 711, 712, 713, 714, 715, 716,717, 718, 719, 720, 721, 722, 723, 724, 725, 726, 727, 728, 729, 730,731, 732, 733, 734, 735, 736, 737, 738, 739, 740, 741, 742, 743, 744,745, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756, 757, 758,759, 760, 761, 762, 763, 764, 765, 766, 767, 768, 769, 770, 771, 772,773, 774, 775, 776, 777, 778, 779, 780, 781, 782, 783, 784, 785, 786,787, 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799,likewise particularly advantageously 800%, 801, 802, 803, 804, 805, 806,807, 808, 809, 810, 811, 812, 813, 814, 815, 816, 817, 818, 819, 820,821, 822, 823, 824, 825, 826, 827, 828, 829, 830, 831, 832, 833, 834,835, 836, 837, 838, 839, 840, 841, 842, 843, 844, 845, 846, 847, 848,849, 850, 851, 852, 853, 854, 855, 856, 857, 858, 859, 860, 861, 862,863, 864, 865, 866, 867, 868, 869, 870, 871, 872, 873, 874, 875, 876,877, 878, 879, 880, 881, 882, 883, 884, 885, 886, 887, 888, 889, 890,891, 892, 893, 894, 895, 896, 897, 898, 899, likewise particularlyadvantageously 900%, 901, 902, 903, 904, 905, 906, 907, 908, 909, 910,911, 912, 913, 914, 915, 916, 917, 918, 919, 920, 921, 922, 923, 924,925, 926, 927, 928, 929, 930, 931, 932, 933, 934, 935, 936, 937, 938,939, 940, 941, 942, 943, 944, 945, 946, 947, 948, 949, 950, 951, 952,953, 954, 955, 956, 957, 958, 959, 960, 961, 962, 963, 964, 965, 966,967, 968, 969, 970, 971, 972, 973, 974, 975, 976, 977, 978, 979, 980,981, 982, 983, 984, 985, 986, 987, 988, 989, 990, 991, 992, 993, 994,995, 996, 997, 998, 999, or even by 1000%, or even by 10000% or even byup to 100000% or 1000000%, wherein any intermediate value is possible.This applies in particular, but not exclusively, to an increasedaffinity with the high-affinity site of the A beta (monomer, oligomer,fibrils and so forth).

This is indicated by a correspondingly reduced K_(D) value. Compared toa linear, binding peptide having a negative charge at the C-terminus,the K_(D) value, as a measure of the binding affinity of the peptidemodified according to the invention to amyloid beta species, and inparticular to amyloid beta oligomers, is reduced by 1%, 2, 3, 4, 5, 6,7, 8, 9, in particular 10%, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75,76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,94, 95, 96, 97, 98, 99, in particular 99.1, 99.2, 99.3, 99.4, 99.5%,99.6, 99.7, 99.8, 99.9 to 99.99, or even 99.999%, wherein anyintermediate value is possible.

Advantageously, these lower K_(D) values refer in particular, but notexclusively, to the high-affinity site of A beta species (monomer,oligomer, fibrils, and so forth).

Peptides modified according to the invention can therefore be used moreefficiently as probes for diagnostic purposes than linear, bindingpeptides having a negative charge at the free C-terminus, in particularmore efficiently than the linear peptide analogs thereof having anidentical amino acid sequence.

However, they can in particular also be used more efficiently astherapeutic agents than linear, binding peptides having a negativecharge at the free C-terminus, and in particular more efficiently thanthe linear peptide analogs thereof having an identical amino acidsequence.

In the direct comparison of a peptide modified according to theinvention to a peptide having a negative charge at the C-terminus, thepeptide according to the invention performs better in terms of affinityand effectiveness.

The reason is that, within the scope of the invention, it was furtherrecognized that the peptides modified according to the inventionadditionally prevent the formation of particularly toxic amyloid betaoligomers, or cause the destruction and/or final toxification thereof,with higher effectiveness or efficiency than peptides having a negativecharge at the free C-terminus, in particular than the peptide analogsthereof having an identical amino acid sequence. This effectiveness isin particular increased by 1%, 2, 3, 4, 5, 6, 7, 8, 9, in particular10%, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81,82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,99.9, and particularly advantageously even by 100%.

For this purpose, a sample comprising different Aß conformers isfractionated, for example, in the simplest case as an experiment. Ineach fraction, different conformers, such as monomers, oligomers,fibrils or higher aggregates, are enriched corresponding to thefractionating step and can then be exactly determined.

The expression “exactly determined” comprises a calibration step duringthe fractionating with molecules of a known type and behavior. Afterfractionating, only a certain type of conformers of the Aß is present ineach fraction, such as monomers, oligomers or fibrils, and so forth.

For example, the conformers are separated according to the s value orsedimentation coefficient thereof in density gradient centrifugation,serving as the fractionating step. Molecules of differing sizes can havean identical hydrodynamic radius, but nonetheless have different svalues and are separated according to these. Calibration using moleculeshaving a known s value allows the Aß conformers obtained by way ofdensity gradient centrifugation to be exactly determined according tothe s value thereof.

Thereafter, the resultant fractions are treated with and without anactive ingredient and determined by way of RP-HPLC, for example. In thisway, it is possible to determine the effectiveness of the activeingredient.

A further method is described hereafter. The so-called QIAD(quantitative determination of interference with Aß aggregate sizedistribution) test can be used for the quantitative analysis of activeingredients. The method for quantitatively analyzing the influence of anactive ingredient on the particle size distribution of amyloid peptidesand/or proteins in a sample comprises the following steps. First, A betais allowed to aggregate under controlled conditions, whereby different Abeta aggregates develop. The conditions are selected so that aparticularly high number of small, especially cytotoxic A beta oligomersare formed. Next, the substance to be examined, such as one of themodified peptides according to the invention, is added to the sample.The active ingredient changes the particle size distribution in thesample. This change is established quantitatively. The change is ameasure of the reduction, or even of the complete elimination, ofcertain toxic species of a certain particle size. The QIAD method isused to measure the increase or the decrease in A beta aggregates havinga certain particle size. While some A beta aggregates having a certainsize were initially present in the sample, these are reduced, or evencompletely eliminated, under the influence of the active ingredient.Other particle sizes increase or remain constant under the influence ofthe active ingredient. The particles that are formed from the A beta arepreferably separated from each other according to the hydrodynamicradius of the particles. In this way, advantageously a multitude offractions are obtained from the sample. The particles of amyloidpeptides and/or proteins having a certain aggregate size are enriched inthe fractions. This separation of the particles can be carried out byway of density gradient centrifugation. The fractions are spatiallyseparated from each other, such as by way of pipetting them off.Finally, the concentration of A beta in the respective fraction isdetermined by completely denaturing the A beta species during a reversephase (RP-) HPLC carried out subsequent to the fractionating. Thedenaturing of the aggregates can take place completely, for exampleusing 30% acetonitrile and 0.1% trifluoroacetic acid at a columntemperature of 80° C., and separating according to hydrophobicity on aC8 column. Eluting A beta is detected by way of UV absorption at 215 nm.The peak area integration can be performed with Agilent ChemStationsoftware. By considering the resultant values in the computation with apreviously conducted calibration, it is possible to calculate theconcentration of A beta present in the particular fraction. Depending onthe fraction, the mean value from multiple, for example six, experimentsconducted independently of one another can be calculated with theresulting standard deviation. The advantage of HPLC analysis is thatdetection is very sensitive (such as approximately 20 nM or 1.8 ngAß1-42) and quantification is reliable independently of the state ofaggregation and the solvent. A decisive advantage of the method lies inthe coupling of density gradient centrifugation and reverse phase HLPC,which allows also Aß oligomers to be reliably quantified.

The effect according to the invention of increased effectiveness in theelimination (or the formation) of amyloid beta species, and inparticular amyloid beta oligomers, can take place with one of thesemethods, but not exclusively with these methods.

In a particularly preferred embodiment of the invention, the describedeffects of increased affinity and effectiveness of elimination, anddetoxification (or formation) also take place in vitro and/or in vivo.

The invention further relates to a peptide according to the inventionfor binding to A beta peptides.

The invention moreover relates to a method for producing the peptideaccording to the invention by way of peptide synthesis, as known to aperson skilled in the art, for example, organic synthesis methods forarbitrary low-molecular-weight compounds and/or mutagenesis andrecombinant production.

The invention also relates to a composition containing the peptideaccording to the invention, in particular for treating Alzheimer'sdisease.

The present invention further relates to a composition containing thepeptide according to the invention, in particular for preventing toxic Abeta oligomers, or for destroying polymers or fibrils formed thereof.

The “composition” according to the invention can be a vaccine, a drug(such as in tablet form), an injection solution, a food or dietarysupplement, for example, containing the peptide according to theinvention in a formulation to be produced based on expert knowledge.

The invention further relates to a kit containing the peptide accordingto the invention. In such a kit, the peptides according to the inventioncan be packaged in containers, optionally with/in buffers or solutions.All components of the kit may be packaged in the same container orseparately from each other. The kit can moreover include instructionsfor the use thereof. Such a kit can include, for example, the accordingto the invention in an injection vial having a stopper and/or septum. Adisposable syringe can also be included therein, for example.

The present invention further relates to the use of the peptideaccording to the invention as a probe for identifying and qualitativelyand/or quantitatively determining amyloid beta oligomers or fibrils.

The present invention further relates to a probe, containing the peptideaccording to the invention for identifying and qualitatively and/orquantitatively determining amyloid beta oligomers.

Such probes are of great importance in enabling early diagnosis of AD.Early diagnosis allows the disease to be counteracted at a very earlystage.

Such molecular probes contain the polymer according to the invention andoptionally dyes, fluorescent dyes, radioactive isotopes (PET and thelike), gadolinium (MRI), and alternative substances suitable for theimaging of probes and can be injected in the patient, for exampleintravenously. After passing the blood-brain barrier, the probes canbind to A beta oligomers and/or plaque. The A beta oligomers and/orplaque thus marked can be rendered visible using imaging processes, suchas SPECT, PET, CT, MRI, proton MR spectroscopy and so forth.

The invention further relates to the use of the peptide for preventingamyloid beta oligomers and/or amyloid beta peptide aggregates and/oramyloid beta fibrils.

The peptide according to the invention is also used to detoxify toxicamyloid beta oligomers and/or aggregates. It is used in particular tobind to amyloid beta oligomers and/or aggregates and thus formamorphous, non-toxic aggregates.

It was found that, when Aß oligomers are already present, the goal of atreatment must be to address these with substances that have the highestpossible affinity to A beta. De facto, the affinity cannot be highenough, and the corresponding dissociation constant of the peptideaccording to the invention is then in the sub-μM range, better even inthe pM range or even lower.

It was found within the scope of the invention that A beta monomers, asbuilding blocks of the A beta oligomers, are continuously created in thehuman body and presumably are not toxic per se. There is even thepossibility that monomers have a positive function. A beta monomers areable to randomly agglomerate as a function of the concentration thereof.The concentration is dependent on the formation and decomposition ratethereof. If the concentration of A beta monomers in the body rises withincreasing age, spontaneous agglomeration of the monomers to form A betaoligomers is increasingly likely. It is possible that the A betaoligomers thus created multiply analogously to the prions and ultimatelylead to Alzheimer's disease.

It was furthermore found that an important difference between theprevention and treatment of, or even a cure for, AD lies in the factthat prevention can potentially already be achieved by preventing theformation of the first A beta oligomers. Just a few A beta ligands aresufficient for this purpose, which have low affinity and are selectivewith respect to the A beta oligomers.

The formation of the A beta oligomers from multiple monomers is areaction of a high order and is thus dependent to a high degree on the Abeta monomer concentration. As a result, a small reduction in the activeA beta monomer concentration thus already prevents the initial A betaoligomers from forming. The treatment concepts and substances previouslyin development, which were of a more preventive nature, are presumablybased on this mechanism.

However, an entirely different situation must be assumed for thetreatment of AD. A beta oligomers, or potentially even larger polymersor fibrils, are already present then, which multiply by way ofprion-like mechanisms. This multiplication, however, is a reaction of alower order and is then almost independent of the A beta monomerconcentration.

If A beta oligomers have already been created, the goal of a treatmentmust be to address these with substances that have the highest possibleaffinity to Aß oligomers and/or to eliminate these particularlyefficiently and/or to prevent formation particularly efficiently ordetoxify the same. The corresponding dissociation constant would have tobe in the sub-μM, nM or pM range, or even lower.

These requirements in regard to the diagnosis (probes) and treatment ofAlzheimer's disease are met with the provision of the peptides accordingto the invention. The peptides according to the invention, in the spiritof the diagnosis and/or treatment, bind the amyloid beta species, and inparticular the A beta oligomers, with an accordingly low dissociationconstant. The invention accordingly further relates to the use of thepeptides according to the invention as a therapeutic agent forAlzheimer's disease.

The peptides according to the invention bind particularly well to A betaoligomers, and in particular to soluble A beta oligomers.

A high specificity for and/or affinity to the target molecule of thepeptides according to the invention causes particularly strong bindingof the peptides according to the invention to the target molecules. Theformed complexes thus have a lower dissociation constant (KD value).

Using the thioflavin T assay, it was possible to show that the peptidesaccording to the invention very efficiently inhibit the fibril formationof A beta peptides, especially the peptides of SEQ ID NOS: 1-12, inparticular SEQ ID NO: 4, SEQ ID NO:5, SEQ ID NO: 6, SEQ ID NO: 7 and/orSEQ ID NO: 8.

The invention further relates to the use of the peptides according tothe invention in a method for treating (in vitro, ex vivo) blood, bloodproducts and/or organs, characterized in that the blood, the bloodproducts and/or the organs are derived from the human or animal body andA (amyloid) beta oligomers are removed and/or detoxified.

The invention will be described in more detail hereafter based onexemplary embodiments and the accompanying figures, without therebylimiting the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Object recognition test

FIG. 2: RotaRod

FIG. 3: SHIRPA

FIG. 4: Morris water maze

FIG. 5: Object recognition

FIG. 6: K_(D) values (kinetics) for amyloid beta monomer;

FIG. 7: K_(D) values (kinetics) for amyloid beta oligomer; and

FIG. 8: K_(D) values (kinetics) for amyloid beta fibrils.

DETAILED DESCRIPTION OF THE INVENTION

The QIAD (quantitative determination of interference with Aß aggregatesize distribution) test was used for the quantitative analysis of activeingredients. The method for quantitatively analyzing the influence of anactive ingredient on the particle size distribution of amyloid peptidesand/or proteins in a sample comprises the following steps. First, A betais allowed to aggregate under controlled conditions, whereby different Abeta aggregates develop. For the result summarized in Table 1, theconditions were selected so that a particularly high number of small,especially cytotoxic A beta oligomers were formed. Next, the substanceto be examined, such as one of the aforementioned D-enantiomericpeptides, is added to the sample. The active ingredient changes theparticle size distribution in the sample. This change is establishedquantitatively. The change is a measure of the reduction, or even of thecomplete elimination, of certain toxic species of a certain particlesize. The QIAD method is thus used to measure the increase or thedecrease in A beta aggregates having a certain particle size. While someA beta aggregates having a certain size were initially present in thesample, these are reduced, or even completely eliminated, under theinfluence of the active ingredient. Other particle sizes increase orremain constant under the influence of the active ingredient. Theparticles that are formed from the A beta are preferably separated fromeach other according to the hydrodynamic radius of the particles. Inthis way, advantageously a multitude of fractions are obtained from thesample. The particles of amyloid peptides and/or proteins having acertain aggregate size are enriched in the fractions. This separation ofthe particles can be carried out by way of density gradientcentrifugation. The fractions are spatially separated from each other,such as by way of pipetting them off. Finally, the concentration of Abeta in the respective fraction is determined by completely denaturingthe A beta species during a reverse phase (RP-)HPLC carried outsubsequent to the fractionating. The denaturing of the aggregates cantake place completely, for example using 30% acetonitrile and 0.1%trifluoroacetic acid at a column temperature of 80° C., and separatingaccording to hydrophobicity on a C8 column. Eluting A beta is detectedby way of UV absorption at 215 nm. The peak area integration can beperformed with Agilent ChemStation software. By considering theresultant values in the computation with the previously conductedcalibration, it is possible to calculate the concentration of A betapresent in the particular fraction. Depending on the fraction, the meanvalue from multiple, for example six, experiments conductedindependently of one another should be calculated with the resultingstandard deviation. The advantage of HPLC analysis is that detection isvery sensitive (such as approximately 20 nM or 1.8 ng Aß1-42) andquantification is reliable independently of the state of aggregation anda solvent. A decisive advantage of the method lies in the coupling ofdensity gradient centrifugation and reverse phase HLPC, which allowsalso Aß oligomers to be reliably quantified.

The results are summarized in Table 1. They show that the substanceseliminate oligomers particularly efficiently.

TABLE 1 QIAD: reduction of Substance/Peptide oligomers in % 1 D3amidated 56 2 RD2 amidated 78 3 D3D3 amidated 98 4 RD2RD2 amidated 97 5RD2D3 amidated 100 6 D3RD2 amidated 88

Each of the substances was used in a concentration of 32 μg/ml.

The results furthermore show that the tested dimers according to theinvention have a synergistic effect compared to the monomers used at thesame dosage.

Hereafter, several in vivo data for the employed peptides will bepresented, which demonstrate the efficacy of the polymers according tothe invention, in particular that of the dimers, and in particular thatof the dimers according to the invention according to SEQ ID NOS: 4-8.

In animal experiments conducted on various transgenic mouse models(Table 2), it was shown that all previously tested amidated peptides areactive in vivo.

FIG. 1: Here, the result of the so-called “object recognition test”(ORT) is shown. Animals treated with D3D3 (amidated) (details in Table2) and animals treated with placebo were placed individually andseparately from each other in a box in which two objects were located.After the animals had time to explore both objects, the animals wereremoved from the box again and not placed back in there until 24 hourslater. Before that, however, one object was replaced with a new objectdifferent therefrom. Animals having memory now preferably explore thenew object. Animals having no memory of having stayed in the box 24hours earlier spend the same amount of time exploring the old object andthe new object. The time that the animal spends with the old object(white bars) and the new object (black bars) is measured in seconds. Itis clearly apparent that the animals treated with the placebo spend thesame amount of time with the new and the old objects. Animals treatedwith D3D3, however, spend much more time with the new object. Thus, onlythe animals treated with D3D3 show a functioning memory in the test.

FIG. 2: The so-called “rotarod” test is used to measure how long ananimal is able to balance on a rotating rod before falling off. Thisallows the animal's neuromotor performance to be measured. The longerthe animal is able to balance, the less strongly the motor neurons areaffected by the phenotype of neurodegeneration. The measurement wascarried out prior to and after the treatment (treatment details areprovided in Table 2). The difference between before and after treatmentis plotted in seconds. The positive value for the control group (blackbar) indicates that the untreated animals exhibited less degenerationprior to treatment than after the treatment with the placebo. Asexpected, the degeneration has thus progressed. The animals treated withD3 (amidated) and D3D3 (amidated), however, exhibited no worsening ofthe neuromotor symptoms. Thus, the treatment slowed, or even stopped,the neurodegeneration in both instances.

FIG. 3: The SHIRPA test is a test where a number of characteristics,including reflexes, are tested. Each characteristic is assessedseparately from the others, and points are awarded in each case. Thehigher the number of points, the more progressed the phenotype, which isto say the neurological damage, is. The SHIRPA test was measured priorto (black bars) and after (white bards) the treatment (treatment detailsare provided in Table 2). It is apparent that the animals treated withD3D3 (amidated) showed no significant worsening of the SHIRPA value. Theanimals treated with D3 (amidated) and with the placebo, however, showeda more or less pronounced worsening of the SHIRPA value. D3D3 thus actsmore efficiently than D3.

FIG. 4: The so-called “Morris water maze” is used to measure the spatialmemory of animals. The time is measured in seconds that the animalrequires to locate a platform hidden closely beneath the water surfacein a water tank. This is measured in several experiments per day onseveral consecutive days. A statistical evaluation then shows whetherthe learning in terms of locating the platform was better among thetreated animals compared to the control group. The search time isplotted in seconds on five consecutive days. The learning of animalstreated with RD2D3 (amidated) was significant.

FIG. 5: Here, the result of the so-called “object recognition test”(ORT) is shown. Animals treated with RD2D3 (amidated) (details in Table2) and animals treated with placebo were placed individually andseparately from each other in a box in which two objects were located.After the animals had time to explore both objects, the animals wereremoved from the box again and not placed back in there until 24 hourslater. Before that, however, one object was replaced with a new objectdifferent therefrom. Animals having memory now preferably explore thenew object. Animals having no memory of having stayed in the box 24hours earlier spend the same amount of time exploring the old object andthe new object. The time that the animal spends with the old object(black bars) and the new object (white bars) is measured in seconds. Itis clearly apparent that the animals treated with the placebo spent thesame amount of time with the new and the old objects. Animals treatedwith RD2D3, however, spend much more time with the new object. Thus,only the animals treated with RD2D3 showed a functioning memory in thetest.

The amidated forms of D3 and D3D3 are able to stop the progression ofthe phenotype in TBA 2.1 mice during the treatment period of 4 weeks(see also FIG. 2).

In particular, it was possible to confirm that the double peptide D3D3(amidated) acts more efficiently than the simple peptide D3 (amidated),and more particularly in the SHIRPA test, likewise in the TBA 2.1 animalmodel (FIG. 3). D3D3 was able to demonstrate in the SweDI mouse modelthat it considerably improves cognition (in the object cognition test,FIG. 1) compared to untreated mice. RD2D3 (amidated) is likewise able toimprove the cognition of SweDI mice, which was demonstrated both in theMorris water maze (FIG. 4) and in the object recognition test (FIG. 5).

FURTHER EXEMPLARY EMBODIMENTS

The following steps refer both to affinity studies and to studies on thedecomposition of particularly toxic amyloid beta oligomers.

Production of Aß Monomers, Oligomers, and Fibrils

1 mg lyophilized Aß1-42 and N-terminal biotinylated Aß1-42 were eachdissolved in 1 ml 100% hexafluoroisopropanol (HFIP) and dissolvedovernight at room temperature. For the oligomer and fibril preparation,non-biotinylated Aß was used with N-terminal biotinylated Aß at a ratioof 1:10 and the HFIP was evaporated (Concentrator 5301 from Eppendorf).The resultant Aß film, at a final concentration of 80 μM, was placed insodium phosphate buffer (10 mM, pH 7.4) and incubated (RT, 600 rpm). Theincubation time was 3 h for the oligomer preparation, and 24 h for thefibril preparation. 100% N-terminal biotinylated Aß1-42 withoutincubation was used for the preparation of monomers.

Density Gradient Centrifugation

The density gradient centrifugation was carried out subsequent to the Aßpreparation to purify the respective Aß species according to the size ofthe same. An iodixanol gradient in 10 mM sodium phosphate buffer, pH 7.4with rising concentrations from 50% to 5% v/v iodixanol was used forthis purpose. 100 μl of the Aß sample was applied and separated by wayof ultracentrifugation (3 h, 4° C., 259000 g). Subsequently, thegradient was fractionated into 14 fractions, 140 μl each. Monomeric Aßare present in the first two top fractions, Aß oligomers in fractions 4to 6, and Aß fibrils in fractions 1 to 13.

Immobilization for Surface Plasmon Resonance (SPR) Spectroscopy A T200from Biacore (GE Healthcare) was used for SPR spectroscopy. The Aßspecies purified by way of density gradient centrifugation were directlyimmobilized on a sensor chip (Series S Sensor Chips SA) according to themanufacturer's instructions by way of biotin-streptavidin coupling.1×PBS was used as the running buffer. Loading took place at 25° C. and aflow rate of 5 μl/min. Subsequently, the flow cells were freed fromnon-specifically bound ligand overnight at a steady flow of 30 μl/min.

Binding Kinetics

Binding kinetics were likewise measured by way of SPR spectroscopy usinga T200 device from Biacore (GE Healthcare). The standard conditions are25° C. and a flow rate of 30 μl/min. Different lyophilizates of the Dpeptides were placed in the 1×PBS running buffer and serially diluted.The method used was a “single-cycle” kinetics method, wherein fiveincreasing analyte concentrations were pumped over the immobilized flowcells. The contact times selected, depending on the analyte, were 90 to120 s for association and dissociation and 1800 to 5400 s for the finaldissociation. The sensorgrams were double referenced with the aid of anunloaded flow cell and the running buffer that was used. The bindingcurves were evaluated by way of kinetic fit models (heterogeneousbinding model) using the Biacore T200 evaluation software (version 2.0).

FIGS. 6 to 8 show the results of the binding behavior of the peptidesaccording to the invention (affinity study). The figures show data forthe kinetic evaluation of the binding strength of the various candidatesto amyloid beta monomers (FIG. 6), amyloid beta oligomers (FIG. 7), andamyloid beta fibrils (FIG. 8). The two binding constants that resultwhen fitting a heterogeneous binding model are shown in each case,plotted as white bars and black bars. It is important to remember that alogarithmic scale is shown here, which means that minor differences inthe bar size represent major differences in the dissociation constantK_(D). White bars are the respective low-affinity sites and the bindingstrengths thereof, and black bars show the high-affinity binding sitesand the binding strengths thereof.

It is shown that a homogeneous 1:1 binding model is only sufficient forfitting the binding curves in the case of the linear peptide D3, inwhich the free C-terminus has a negative charge.

It is also shown that the white bars for the low-affinity binding sitesare similarly high in almost all instances of the cyclized peptides thatare used. However, big differences result in the high-affinity bindingsites, which are shown as black bars.

It also becomes apparent that the cyclic cD3r (SEQ ID NO. 9) does verywell among the monomers (FIG. 6), which is to say has particularly highaffinity.

Among the oligomers that are of particular interest, cD3r binds evenmore strongly by two orders of magnitude than the other cyclizedpeptides according to the invention (FIG. 7).

The cD3r also does very well among the fibrils compared to the otherpeptides according to the invention, wherein here, as a peculiarity, thepeptide according to the invention cD3(P2K) (SEQ ID NO. 12) doesextremely well and advances as far as the sub-pM binding range (FIG. 8).

It is also shown that the amidated form of the D3 binds more strongly toamyloid beta monomer and amyloid beta oligomer than the non-amidatedform of the D3. This is evident from the lower K_(D1) value for thelow-affinity site and the presence of a high-affinity site, which has aK_(D2) in the sub-μ range.

The R_(max) values in FIGS. 6 to 8 indicate how strong the contributionof the respective K_(D) to the overall loading capacity is. In theexample of the oligomers (FIG. 7), an R_(max) of 79/21% is shown for thepeptide cD3z. cD3z denotes the cD3 zero, which is to say cyclized D3without any further amino acid attachments. The white bar indicates thatthe low-affinity site can yield a total of 79% of the RU total loadingstrength and K_(D2) a total of 21% of the RU. This means that a ratio ofapproximately 1:4 exists for this peptide at binding sites, which is tosay there are approximately 4 times as many low-affinity sites ashigh-affinity sites.

Such differences between the low-affinity site and the high-affinitysite occur with the peptides according to the invention.

The observation that no high-affinity site is yielded for the linearpeptide D3 when fitting the experimental binding data can be explainedby the fact that it either does not bind to the high-affinity site, orthat the affinities to the high-affinity site and low-affinity sitecannot be distinguished.

It becomes apparent from this data that the linear D3 binds only alow-affinity site, but not a high-affinity site in the case of amyloidbeta oligomer.

TABLE 2 Experiment Mouse Object Plaque number Peptide model Dose Watermaze recognition pathology SHIRPA Rotarod 1 D3 amidated TBA 0.1 mg/daynot carried not carried not carried significant out out out (FIG. 2) 2D3D3 TgSwDI 1 mg/4 significant significant significant not carried notcarried amidated weeks day 5 (FIG. 1) out out 3 D3D3 TBA 0.1 mg/day notcarried not carried not carried significant significant amidated out outout (FIG. 3) (FIG. 2) 4 RD2D3 TgSwDI 7 mg/4 significant significant notcarried not carried amidated weeks day 5 (FIG. (FIG. 5) out out 4) Eachof the substance was administered i.p. for 4 weeks (micropump).

The invention claimed is:
 1. A peptide comprising an amino acid sequencewhich binds to amyloid beta species, wherein the peptide has beenmodified by cyclization, wherein the amino acid sequence is selectedfrom the group consisting of SEQ ID NO: 1, SEQ ID NO: 9, SEQ ID NO: 10,SEQ ID NO: 11 and homologs thereof, with an identity of at least 80%,and wherein the peptide's binding affinity to the amyloid beta speciesis increased as compared to an analogous peptide without anymodification.
 2. The peptide according to claim 1, whereinD-enantiomeric amino acids make up at least 50% of the amino acidsequence.
 3. The peptide according to claim 1, being linked to a furthersubstance.
 4. The peptide according to claim 1, comprising at least twoamino acid sequences, one of which is SEQ ID NO: 1 and the other beingselected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ IDNO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 and homologs thereof.5. The peptide according to claim 4, wherein the at least two amino acidsequences together form a second amino acid sequence selected from thegroup consisting of SEQ ID NO: 4 and SEQ ID NO: 5, and homologs thereof.6. A method for producing a peptide according to claim 1, comprising astep of producing the peptide by way of peptide synthesis ormutagenesis.
 7. A kit comprising a peptide according to claim
 1. 8. Acomposition comprising a peptide according to claim 1.